The long-term objective of this project is to determine how the clinical problem of ischemia-reperfusion (I/R) induces fluid filtration from the microvasculature, and also how such microvascular dysfunction is exacerbated in the presence of the cardiovascular risk factor hypercholesterolemia. I/R-induced tissue injury is thought to be mediated by neutrophil-endothelial cell interactions and nitric oxide suppression, and by agents such as oxygen radicals and proteolytic enzymes. Investigation on the effects of these mediators on the venular endothelium is well underway, but knowledge is scarce on how they affect capillary endothelium, either in normal individuals or those with hypercholesterolemia. In the proposed studies, rats will be fed either a normal or high cholesterol diet, so that the effect of this cardiovascular risk can be evaluated in postischemic capillaries. Ischemia of mesenteric vessels will be produced by ligating the superior mesenteric artery for various durations. Prior to and following ischemia, measurements of filtration will be made by occluding a selected capillary with a micropipette, and determining the fluid filtration rate (Jv/S) from the decreasing distance between intravascular red blood cells upstream of the occlusion site. Preliminary experiments demonstrate that hypercholesterolemia enhances I/R-induced Jv/S and that neutrophils are primarily responsible. The first of three specific aims of the proposed project is to establish the time course of I/R-induced increases in Jv/S and to determine the extent to which hypercholesterolemia modifies this filtration response. The second specific aim is to determine the mode of action whereby neutrophils are involved in hypercholesterolemia-enhanced increases in Jv/S following I/R. Possibilities include 1) direct interaction with the capillary endothelium, 2) an upstream propagated response due to postcapillary adhesion, 3) diffusion of neutrophil-derived products through interstitial fluid, and 4) recirculation of neutrophil-derived products through the bloodstream. The third specific aim is to determine whether adhesion molecules, oxidants, proteases, and nitric oxide suppression are included in the neutrophil-mediated increase in Jv/S associated with hypercholesterolemia following I/R. Identification of the mediators of the enhanced capillary filtration elicited by I/R may help in developing therapeutic strategies to reduce the higher mortality rates associated with risk factors of ischemic heart disease.